The invention relates to a method of preparing a prothrombin complex, a solution of serum proteins of good stability in storage, and a concentrate containing antihemophilic globulin A, on the basis of a special starting material.
The great need for human proteins makes it necessary to process human plasma by a method whereby all of the plasma proteins will be obtainable by fractionation with a minimum of loss. The method known as "Cohn fractionation" (Cohn E. J., Gurd F. R. N., Surgenor D. M., Barnes B. A., Brown R. K., Deronaux G., Gillespie J. M., Kahnt F. W., Lever W. F., Lin C. H., Mittelmann D., Monton R. R., Schmid K. and Uroma E.: "A system for the separation of the components of human blood: Quantitative procedure for the separation of the protein components of human plasma" J. Amer. Chem. Soc. 72 (1950), p. 465) is well established, although many plasma proteins are so altered or denatured by this process that they are no longer suitable for administration by infusion, or else they are completely lost. The yields of the desired products, therefore, are very low. Furthermore, the gamma globulin present in the starting material is destroyed.
In the modified Cohn fractionation most frequently used today (Kistler P. and Nitschmann H., "Large Scale Production of Human Plasma Fractions," Vox Sang., 7, (1962), pp. 414-424), only three products are obtained from the plasma, namely albumin, gamma globulin, and coagulable fibrinogen. Due to the danger of hepatitis, the fraction I containing the fibrinogen is made or can be made only from small plasma pools. The alcohol which is used in these fractionations, being an organic solvent, has a denaturing effect on the plasma proteins (Kauzmann W., Avan. Protein Chem., 14 (1959), p 37). The compounds between the hydrophobic protein radicals, which are important to the structure and stability of the proteins, are broken up by the alcohol used in the fractionation, resulting in the denaturation of the proteins and also in the formation of protein aggregations. Such aggregated and denatured proteins are present, for example, in standard gamma globulin preparations unsuitable for intravenous use. Consequently, attempts have been made to isolate proteins without the use of precipitants.
It is known that, after the thawing of citrate plasma at 0.degree. to 8.degree. C after it has been frozen, a small amount of a protein precipitate remains which contains factor VIII activity known as antihemophilic globulin A. The factor VIII content of this fraction is great enough to permit clinically useful factor VIII concentrate to be made from it, the disadvantage, however, being that the blood that is to be processed for cryoprecipitate has to be stabilized with citrate, so that the citrate-containing plasmas then must be processed by the inadequate Cohn method, or else the citrate has to be removed from the plasma by dialysis, which as yet cannot be accomplished on a large technical scale.
Factor IX, together with factors II, VII and X, is part of the so-called "prothrombin complex" or "vitamin K-dependent coagulation factors," all of which are synthesized in the liver. All of the methods for the purification of the prothrombin complex rely on the fact that these factors can be adsorbed onto inorganic adsorbents or onto ion exchange cellulose. In order for the prothrombin complex to be adsorbed directly from plasma onto barium sulfate or calcium phosphate, the blood has to be taken in special anticoagulants, in which case the cell components are no longer usable for transfusion. Barium sulfate yields toxic end products (Tullis J. L., Melin M. and Jurigian P., "Clinical use of human prothrombin complexes," New England Journal of Medicine 273 (1965), 667), and therefore cannot be used.
A method is also known in which a prothrombin complex concentrate can be prepared from plasmas stabilized with citrate, from which the cryoprecipitate has already been removed. This process, nevertheless, has two decided disadvantages:
1. The process is unsuitable for large amounts of plasma. Only small plasma pools can be processed, since the citrate has to be removed by dialysis so that the prothrombin complex will be susceptible of adsorption and elution. PA0 2. The aluminum hydroxide that is used can, in the presence of citrate, result in increased concentration of aluminum due to chelation.
Lastly, a method has also been described for obtaining the prothrombin complex from ACD plasma (citric aciddextrose plasma) by the use of diethylaminoethyl cellulose. In this procedure, however, the plasma must be diluted with an equal volume of water for the purpose of diluting the citrate concentrations prior to adsorption. This creates considerable difficulty in the further fractionation. Consequently, appreciable disadvantages encumber all of the known methods for the production of the prothrombin complex.
The limited usefulness of blood stabilized with anticoagulants appears also in the production of cryoprecipitate. Since in the production of Factor VIII it is desirable to process all possible plasmaphoresis plasmas to yield cryoprecipitate, it has been virtually obligatory to use citrate as the stabilizer.